Abstract The striatum is a subcortical brain region critical for action selection, learning, and motor behavior. Striatal cholinergic interneurons (ChIs) are the primary source of local acetylcholine, and play an important role in modulating striatal circuits. Consequently, ChIs are dysregulated in many neuropsychiatric disorders that involve striatal dysfunction, such as Parkinson's disease, Alzheimer's disease, and dystonia. Despite their importance, little is known about ChI structure and function. ChIs are a sparse cell type, comprising only ~1% of all striatal neurons, yet have tremendous territorial influence by sending out dense projections. The complete morphology and local network anatomy of ChIs remain unknown. Furthermore, a recent study suggested ChIs are not homogenous as previously believed, but may be divided into subtypes based on morphology and molecular markers. However, the correlation between ChI anatomy and molecular profiles has yet to be elucidated. This proposal seeks to expand our understanding of ChI anatomy and molecular profiles by developing new tools to answer questions that were impossible to address before. Although previously understood to be an aspiny, homogenous population, my preliminary data demonstrates the existence of a new ChI morphological subtype: spiny ChIs. I will characterize the complete morphologies of spiny and aspiny ChIs by combining Brainbow with new clearing and expansion technology. I will then develop technology to simultaneously visualize morphology and interrogate the molecular profile of ChIs in order to correlate the two. These studies will increase our understanding of ChI cell types, and reveal fundamental principles of how their anatomy and molecular profiles contribute to function. The novel tools developed in this project will also be disseminated for the benefit of the neuroscience community. Finally, completing this proposal will help drive me towards my long-term goal of becoming a physician scientist by providing me with rigorous scientific, technical, and clinical training.